Tonic protein kinase A activity maintains inactive beta2 integrins in unstimulated neutrophils by reducing myosin light-chain phosphorylation: role of myosin light-chain kinase and Rho kinase.

Activation of beta2 integrins is necessary for neutrophil adhesion and full activation of neutrophil effector functions. We demonstrated previously that inhibition of protein kinase A (PKA) activity in quiescent neutrophils is sufficient to increase beta2-integrin cell surface expression, affinity, and adhesion. Thus, a tonic level of PKA activity prevents inappropriate activation of beta2 integrins in unstimulated neutrophils. Myosin light-chain (MLC) phosphorylation is an important regulator of leukocyte integrin function and adhesion. Moreover, PKA regulates MLC phosphorylation via inhibiting MLC kinase (MLCK) and MLC dephosphorylation via effects on the Rho kinase (ROCK)/MLC phosphatase pathway. We hypothesize that the tonic inhibitory effect of PKA on beta2-integrin activation neutrophils operates via its inhibition of MLC phosphorylation. We demonstrate here that inhibition of PKA activity with KT5720 activated beta2 integrins and adhesion coincident with an increase in MLC serine 19 (Ser 19) phosphorylation. KT5720-induced activation of beta2 integrins, adhesion, and MLC Ser 19 phosphorylation was abolished by pretreatment with the MLCK inhibitor ML-7 and specific MLCK inhibitory peptides but not the ROCK inhibitor Y-27632. These findings demonstrate that tonic PKA activity prevents activation of beta2 integrins and adhesion by inhibiting MLC phosphorylation via a MLCK-dependent but ROCK-independent pathway.

Randomized, controlled study of inhaled fluticasone propionate, oral administration of prednisone, and environmental management of horses with recurrent airway obstruction.

OBJECTIVE: To determine whether administration of glucocorticoids provides additional benefits to environmental management of horses with recurrent airway obstruction (RAO). ANIMALS: 28 horses with RAO. PROCEDURE: Horses were classified as having mild, moderate, or severe RAO. Within each category, horses were randomly assigned to receive inhaled fluticasone propionate, inhaled control substance, or oral administration of prednisone. During the 4-week study, horses were maintained outdoors and fed a pelleted feed. Clinical scores, pulmonary function, results of cytologic examination of bronchoalveolar lavage fluid (BALF), and adrenal gland function were determined before and 2 and 4 weeks after initiation of treatment. RESULTS: Clinical score and pulmonary function of all RAO-affected horses improved during the treatment period. After 4 weeks, clinical scores and pulmonary function of horses treated with a glucocorticoid were not different from those for the control treatment. In horses with severe RAO, treatment with fluticasone for 2 weeks resulted in significantly greater improvement in pulmonary function, compared with pulmonary function after treatment with prednisone or the control substance. Treatment with a glucocorticoid for 4 weeks and a low-dust environment did not have any effect on cellular content of BALF Treatment with prednisone for 2 weeks resulted in a significant decrease in serum cortisol concentration, compared with concentrations after administration of fluticasone or the control substance. CONCLUSIONS AND CLINICAL RELEVANCE: Environmental management is the most important factor in the treatment of horses with RAO. Early treatment with inhaled fluticasone can help accelerate recovery of horses with severe RAO.

The effects of cAMP modulation upon the adhesion and respiratory burst activity of immune complex-stimulated equine neutrophils.

Toxic products such as reactive oxygen intermediates released by activated polymorphonuclear neutrophil (PMN) have an important role in the pathophysiology of diseases associated with the deposition of immune complexes (IC) in tissues. IC-induced activation of PMN requires adhesion mediated by integrin adhesion receptors. Of the integrins expressed on PMN, the beta(2) family has been found to be of particular importance for activation of PMN by IC. beta(2) Integrin ligand binding must be activated to enable adhesion to IC. Both activating and inhibitory signals regulate beta(2) integrin ligand avidity and adhesion. The second messenger cyclic adenosine monophosphate (cAMP) has been demonstrated to inhibit the activation of PMN in response to a variety of stimuli. The purpose of this study is to test the hypothesis that cAMP-dependent signals inhibit beta(2) integrin-dependent adhesion of equine PMN to immobilized IC and subsequent adhesion-dependent activation of respiratory burst activity. Treatment of equine PMN with beta(2) adrenergic agonists isoproterenol or clenbuterol, which trigger an increase in intracellular cAMP concentration, inhibited adhesion of equine PMN to IC in a dose dependent manner. Similarly, inhibition of cAMP hydrolysis by the non-specific phosphodiesterase (PDE) inhibitor pentoxifylline and the PDE 4-specific inhibitor rolipram inhibited adhesion of equine PMN to IC. Elevation of intracellular cAMP levels with pentoxifylline, clenbuterol and rolipram also inhibited IC-induced activation of respiratory burst activity in equine PMN. Importantly, co-treatment of equine PMN with rolipram and either beta(2) adrenergic agonist synergistically inhibited both the adhesion of equine PMN to IC as well as the subsequent respiratory burst activity.